Volume 32, Issue 5, Pages 703-713 (May 2010) Immobilized Chemokine Fields and Soluble Chemokine Gradients Cooperatively Shape Migration Patterns of Dendritic Cells  Kathrin Schumann, Tim Lämmermann, Markus Bruckner, Daniel F. Legler, Julien Polleux, Joachim P. Spatz, Gerold Schuler, Reinhold Förster, Manfred B. Lutz, Lydia Sorokin, Michael Sixt  Immunity  Volume 32, Issue 5, Pages 703-713 (May 2010) DOI: 10.1016/j.immuni.2010.04.017 Copyright © 2010 Elsevier Inc. Terms and Conditions

Immunity 2010 32, 703-713DOI: (10.1016/j.immuni.2010.04.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Organized Dendritic Cell Migration on Lymph Node Cryosections (A) Contrast enhanced serial images of mature dendritic cells (DCs) on a section. Margins of the section are highlighted by the dotted line. Areas of high DC density are outlined (black line). Red arrows indicate entry sites of DCs. Small insert: brightfield image of the lymph node (LN) cryosection. Black boxes mark T cell area (T), sinus area (S), and control area (C) used for quantification in (B). Scale bar represents 200 μm. (B) DCs were quantified in the areas C, S, and T by automated density measurement. The upper diagram represents mature; the lower represents immature DCs. Data represent one out of > 30 independent experiments. (C) Composite picture of serial images of (A). Different time points were colorized in graded wavelengths from yellow (1 hr) to red (18 hr) to show migratory fractions of DCs on the section. Dotted line: outline of the section. (D) Left panel: section of a lymph node showing fluorescently labeled DCs (green) entering the lymph node via interfollicular areas 10 hr after subcutaneous injection. Laminin staining (red) highlights the reticular fibers surrounding the B cell follicle. Right panel: single-cell tracking of DCs migrating on a consecutive section of the one shown left. Each track represents the pathway of a single cell. Dotted line: outline of the section. Scale bar represents 50 μm. Immunity 2010 32, 703-713DOI: (10.1016/j.immuni.2010.04.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Dendritic Cells Are Directed by Soluble Gradients (A) Accumulation of wild-type (WT), Ccr7−/−, and plt/plt dendritic cells (DCs) on either WT or plt/plt lymph node sections. Bars represent mean ± SEM, n = 3. (B) Fluorescent staining of a lymph node section for CCL21 (green) and ICAM-1 (red). Scale bar represents 250 μm. (C) A peripheral part of a lymph node section was dissected, inverted by 180°, and repositioned in close proximity to the remaining part of the section. Left panel: bright-field image of the manipulated section. Right panel: Migratory course of mature DCs on this section. Different time points were colorized in graded wavelengths from yellow (1 hr) to red (4 hr) to show migrating fractions of DCs. Dotted line: outline of the section. Data represent one out of four independent experiments. Scale bar represents 200 μm. ∗∗∗p < 0.001; ns, not significant. Immunity 2010 32, 703-713DOI: (10.1016/j.immuni.2010.04.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Migration of Dendritic Cells toward Immobilized CCL21 CCL21 was plated on cell culture plastic (coated area marked by dotted circle). (A) Distribution of dendritic cells (DCs) on CCL21 coated area after 2 min (left panel) and 15 hr (right panel). Data show one representative out of > 30 independent experiments. (B) Ccr7−/− DC distribution after 15 hr. (C) Single-cell tracking of migrating WT DCs. Each track represents the pathway of a single cell over 30 min. Migration is directed toward the CCL21 coated area (arrow) and is observed in uncoated areas up to 1 mm from the coated area. Immunity 2010 32, 703-713DOI: (10.1016/j.immuni.2010.04.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Dendritic Cells Turn Immobilized CCL21 into Soluble CCL21 (A) Number of migrated cells after 15 hr. Ptx, pertussis-toxin; Apr, aprotinin; Sup, cell supernatant; CCL21 trunc., CCL21 with truncated C terminus. If not otherwise indicated, WT DCs were applied. Error bars represent mean ± SEM, n = 4–8. (B) CCL21 was incubated with DCs for up to 12 hr. Processing of CCL21 was determined by immunoblot analysis. Control (−), CCL21 incubated in medium without DCs. (C) DCs, B cells, T cells, and fibroblasts were coincubated with CCL21 for 12 hr before immunoblot analysis of the supernatant. (D) In vitro: CCL21 cleavage after 12 hr coincubation with DCs. In vivo: pull-down of in vivo cleaved CCL21 out of LN-lysate. (E) CCL21 was incubated with DCs or with DCs and Aprotinin for 15 hr. Control (−): CCL21 incubated in medium without DCs. (F) Immunoblot analysis of tagged CCL21 (detection of CCL21, His- and Flag-tag) after 15 hr incubation with DCs (+). Control (−): CCL21-incubation in medium without DCs. ∗∗∗p < 0.001; ns, not significant. Immunity 2010 32, 703-713DOI: (10.1016/j.immuni.2010.04.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Immobilized chemokine causes haptokinetic migration (A) Morphology of a dendritic cell (DC) applied to a lymph node section after 0, 2, and 4 hr. Upper panels: elongated, nondendritic “amoeboid” shape of a DC migrating on a sinus area. Lower panels: rounded, dendritic-like morphology of a nonmigrating DC on a B cell follicle. The cell surfaces are highlighted in green. Scale bar represents 10 μm. (B) Accumulation of WT and Itgb2−/− DCs on a lymph node sections. Error bars represent mean ± SEM, n = 3. (C) Quantification of the 2D projected cell surface after contact with either CCL21 plus ICAM-1, CCL19 plus ICAM-1, or BSA plus ICAM-1 coated glass surfaces after 1, 5, 10, and 15 min. Error bars show mean ± SEM, n = 25. (D) 2D projected surface of DCs on ICAM-1 coated glass surfaces after 1, 5, 10, and 15 min incubation with CCL21, CCL19, or BSA applied to the medium. Error bars represent mean ± SEM, n = 25. (E) 2D projected surface of WT, Ccr7−/−, and Itgb2−/− DCs after contact with a plasma-treated and subsequently CCL21 plus ICAM-1, CCL19 plus ICAM-1, or BSA plus ICAM-1 coated glass surface after 1, 5, 10, and 15 min. Error bars represent mean ± SEM, n = 25. (F) Outline of a DC polarizing on a CCL21 plus ICAM-1-spot over 13 min. The surface of the DC is highlighted in green. Scale bar represents 25 μm. ∗∗∗p < 0.001. Immunity 2010 32, 703-713DOI: (10.1016/j.immuni.2010.04.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Soluble Chemokine Steers Haptokinetic Migration Three-dimensional collagen gel assays with incorporated protein-coated carbon microfibers. (A, E, F, and G) Left panels: bright-field image of the collagen gel with coated fibers. Right panels: single-cell tracking of dendritic cells (DCs). Here, each colored track represents the pathway of a single cell. (A) DCs in the presence of CCL21/ICAM-1-coated carbon fibers. (B) Differential interference contrast image of DCs spreading on a CCL21 plus ICAM-1-coated fiber. Scale bar represents 50 μm. (C) Quantification of WT and Itgb2−/− DCs associated with CCL21 plus ICAM-1-coated fibers after 5 hr incubation. Bars represent mean ± SEM, n = 5. (D) Quantification of WT DCs associated with with CCL21 plus ICAM-1 coated fibers after 5 hr incubation in a collagen gel with different CCL19 gradients applied. Bars represent mean ± SEM, n = 4. (E) Directionally biased DCs migration on CCL21 plus ICAM-1 coated fibers within a CCL19 gradient. DCs remote from coated fibers migrate through the collagen gel toward the CCL19 gradient. (F) WT DCs migrating with a directional bias in a collagen gel with CCL21 plus ICAM-1-coated fibers toward a gradient of truncated CCL21. (G) Itgb2−/− DCs migrating in a collagen gel with CCL21 plus ICAM-1-coated fibers and applied CCL19-gradient. ∗∗∗p < 0.001. Immunity 2010 32, 703-713DOI: (10.1016/j.immuni.2010.04.017) Copyright © 2010 Elsevier Inc. Terms and Conditions